Cells of the innate immune system constantly evaluate host mucosal surfaces and peripheral tissues for signs of infection or injury. The host must find a balance between tolerance of beneficial microorganisms and minor non-pathological microbial encounter vs. the development of a robust immune response to more serious infections. Emerging evidence suggests that this decision is made by the cell based on the combinatorial signals it receives from its engagement with microorganisms and endogenous stimuli. These signals are sensed primarily by various classes of pattern recognition receptors (PRR), and while there has been remarkable progress in characterizing the individual signaling pathways induced through these receptors, relatively few studies have addressed how immune cells integrate combined PRR inputs and the combination of these signals with others arising from soluble host derived substances such as cytokines, lipids, and complement components. This project seeks to define the control principles that determine the complex relationship between signal input and output function in this scenario, and ultimately to generate quantitative computational models to describe cellular behavior in circumstances relevant to infectious disease. To evaluate the macrophage response to combined microbial stimuli, we have profiled the response of murine macrophages to single and pairwise combinations of toll-like receptor (TLR) ligands that induce either one or both of the MyD88 and TRIF branches of the TLR signaling pathway. We find predominantly less than additive levels of mRNA induction in response to pairwise stimuli. This might be expected due to saturation of the common signaling effectors used by TLR pathways; however, it is noteworthy that in some cases (particularly with plasma membrane localized TLRs), the dual ligand phosphoprotein response is lower than with either single ligand, suggesting that in ligand combinations that signal exclusively through the MyD88 adapter, there are additional signal suppression mechanisms at work beyond simple saturation of components. On the other hand, in ligand combinations including a TRIF-activating ligand, a select subset of key immune response mediators are induced to greater than additive levels. We predict that this synergistic response requires signaling through both the TRIF and MyD88-dependent pathways, and that it signals to the host the presence of a dangerous pathogenic challenge. We seek to determine the molecular mechanisms underlying this selective pattern of non-linear macrophage responses to pathogenic ligands, as it would provide important insight to signaling pathway crosstalk during a microbial infection. This year, to address how the TLR signaling network might mediate the suppressed response through combined MyD88 stimuli, we investigated the localization dynamics of proximal signaling components in the TLR pathway. While comparing the localization of these proximal signaling components in response to single vs. combined TLR ligands, we unexpectedly observed that mouse IRAK1 forms aggregate bodies in the cytoplasm upon dual TLR ligand stimulation or with very high concentrations of single ligand. Interestingly, cells enriched in these IRAK1 clusters have low MAP kinase activity and lower nuclear staining of TLR-activated transcription factors like pATF2, consistent with the less than additive responses we observe to dual ligand stimulation of these cells. IRAK1 clusters also colocalize with TLR signaling proteins distal in the pathway to IRAK1, suggesting that IRAK1 may function to sequester these components. Furthermore, in IRAK1KO cells, phosphoprotein and cytokine responses to single TLR ligands are comparable to WT cells, but responses of IRAK1 KO cells to combined TLR ligands are increased. We conclude from these data that contrary to the traditional view of TLR signalling in mouse macrophages, IRAK1 may attenuate TLR responses in mouse macrophages under conditions of high signaling flux, such as when multiple bacterial ligands are presented simultaneously. We also seek to identify the basis of the greater than additive release of two key cytokines, IL-6 and IL-12, from macrophages in response to ligands which induce the TRIF and MyD88-dependent pathways. This likely facilitates detection of combined exposure to viral and bacterial pathogens, or significant infection with an intracellular pathogen, and leads to the increased production of cytokines that serve to drive a robust adaptive immune response. We previously identified approximately 200 genes with expression characteristics that could implicate them as regulators of the enhanced production of IL6 and IL-12. These genes were targeted in an siRNA screen to determine if they are required for the macrophage to induce high levels of these cytokines in response to a combined TLR stimulus. This year, 22 of the most robust hits were subjected to high throughput qPCR analysis to determine if they selectively affect different classes of TLR-induced inflammatory mediators. This analysis has identified both negative and positive regulators of IL-6 and IL-12 p40, and finds that the effects on these cytokines are also observed for additional secondary responses genes, and also class II primary response genes. Many of the identified genes are not well characterized in the literature, and may provide novel therapeutic targets for regulating TLR-driven outputs. To evaluate the TLR signal integration that occurs in the context of a real infection, we previously initiated a study of the macrophage response to Burkholderia cenocepacia (Bcc), an opportunistic bacteria particularly problematic in cystic fibrosis and chronic granulomatous disease patients, and closely related to the category A select agents B. mallei and pseudomallei. Macrophages are likely to play a key role in Bcc-induced pulmonary infections, but very little is known about the mechanism of Bcc infection and replication in these cells. We previously published a comprehensive characterization of the intracellular life cycle of B. cenocepacia and its interaction with the autophagy pathway in human macrophages (Al-Khodor et al. Cell Microbiol. 16(3):378-95). We demonstrated that the invading bacteria interact transiently with the endocytic pathway before escaping to the cytosol. This escape triggers the selective autophagy pathway, however, despite recruitment of all the key autophagy initiation components, B. cenocepacia blocks autophagosome completion and replicates in the host cytosol. This year we took advantage of the pathogenesis characteristics of Bcc to develop high content imaging assays that permit robust quantification of multiple stages of Bcc infection of macrophages (Miller et al. (2015) Assay Drug Dev Technol. In press). Using this assay and imaging platform, we ran a screen for host components that recognize and ubiquitinate cytosolic bacteria, as this is a key initial step in the selective autophagy response to numerous pathogenic microbes. We have identified several host proteins not previously implicated in pathogen responses whose perturbation leads to reduced bacterial ubiquitination and increased replication, implicating them as potential infection restriction factors. We also initiated a study in collaboration with the LSB Cellular Networks Proteomics unit to use mass spectrometry to screen for candidate Bcc effector proteins that could mediate the autophagy subversion described above. Human cells were infected with Bcc and host autophagy pathway effectors were affinity purified and analyzed for associated bacterial proteins. This has identified several bacterial effectors and host proteins as novel candidate regulators of the autophagy process. These studies are ongoing.